The invention generally relates to methods of labeling organic compounds for fluorescent detection. More particularly, the invention relates to rhodamine-based fluorophores which are made useful by derivatization with organic molecules, and have application so as to label biomolecules such as synthetic oligonucleotides and proteins. The fluorophores are single isomers, are stable and reactive in standard phosphite chemistry, and the conjugates remain fluorescent.
The use of fluorescent dyes as detection labels has found widespread application in molecular biology, cell biology and molecular genetics. In particular, the use of fluorescently labeled oligonucleotides has expanded with advancements in DNA sequencing, fluorescence in situ hybridization (FISH), hybridization assays including nucleic acid arrays (xe2x80x9cDNA chipsxe2x80x9d), probe capture assays, fluorescence polarization studies, and DNA amplification assays: polymerase chain reaction (PCR), isothermal amplification assays (strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3SR), and with fluorescent primer and/or probe detection (xe2x80x9cTaqmanxe2x80x9d assay).
Current automated DNA sequencing methods make use of multiple fluorescent labels for concurrent detection of base sequence in a single gel lane or capillary. Most of the commonly used fluorescent dyes for sequencing are produced as a mixture of isomers, including those of the rhodamine family. (In reference to rhodamine dyes, we will use the numbering scheme described in the Colour Index by the Association of Textile Chemists, 2nd Edition, 1971.) Single isomer dye labels are preferred for high resolution techniques such as DNA sequencing and capillary electrophoresis, because slight differences in spectral properties exist between different isomeric forms of the fluorophores. In addition, differences in the electrophoretic mobilities of 5- and 6-isomer fluorophore-tagged primers (e.g. 5- and 6-carboxytetramethylrhodamine) can lead to band broadening if mixtures of isomers are used (Hung et al., Analytical Biochem., 238, 165-170, 1996). Therefore, single isomer forms must be purified before preparing fluorescent dye labeling reagents intended for use in labeling oligonucleotides for DNA sequencing.
Some fluorescent dye labels can be attached to the 5xe2x80x2 end of oligonucleotides during the process of synthesizing the primers (e.g. fluorescein using a fluorescein phosphoramidite reagent). These dye phosphoramidites react properly under phosphite chemistry conditions because protection of the two active oxygen groups on the fluorescein moiety prevent possible side reactions between the phosphoramidite and fluorescein. In addition, modification with the protecting groups holds the 3-position carboxylic acid function in the closed ring lactone form, preventing proton donation from the carboxylate to the N,N-diisopropylamino phosphoramidite. Protonation would convert the diisopropylamino moiety into a good leaving group, which could decompose the reagent. Some rhodamine phosphoramidites synthesized (for example, U.S. Pat. No. 5,231,191, issued Jul. 27, 1993, inventors Woo et al.) have the 3-position carboxylic acid function existing in equilibrium between the closed (lactone) and open (acid) form. When the reagent is used in oligonucleotide synthesis, the xe2x80x9cacidicxe2x80x9d environment will favor formation of the carboxylate-onium cation form. Proton donation from the carboxylic acid moiety to the N,N-diisopropylamino phosphoramidite could occur and result in reagent instability, compromising oligonucleotide labeling efficiency.
Some fluorescent dye labels (e.g. fluorescein and related derivatives) retain their fluorescent properties during cleavage of the labeled oligonucleotide from the solid phase support and removal of protecting groups with concentrated aqueous ammonia, the standard method in current practice. However, dyes in the rhodamine family are susceptible to chemical modification by the ammonia treatment, which drastically decreases their fluorescent properties. Thus, it is a general practice for rhodamine-type dyes to be attached to the 5xe2x80x2 end of oligonucleotides which have been modified with linker functionalities (e.g. primary amine) after automated synthesis, cleavage and deprotection. This dye labeling requires additional steps and manual labor, incurring greater cost and inconvenience in the overall synthesis of 5xe2x80x2-rhodamine dye-labeled oligonucleotides.
In one aspect of the present invention, fluorogenic compounds and compositions are provided that are based upon rhodamine. In rhodamine there is a 3-position carboxylate. Fluorogenic compounds of this invention, or fluorophores, have the 3-position carboxylate converted to a fully substituted amide. One substituent of the amide nitrogen is a group that is effective to block lactam ring formation. The other substituent of the amide nitrogen is useful in making, or includes, desired derivatives. Useful derivatives of these fluorophores can be made for labeling organic compounds for fluorescent detection. Preferred organic compounds are biomolecules such as a peptide, protein, amino acid, nucleotide, oligonucleotide, or nucleic acid polymer. The conjugation is preferably via a phosphoramidite linkage when synthesizing labeled oligonucleotides, and may be by a variety of the known protein conjugation chemistries when synthesizing labeled peptides or labeling.
Formula A below illustrates a rhodamine-based, fluorophore moiety of this invention where Ra and Raxe2x80x2 are both non-hydrogen substituents for the amide nitrogen. 
In the Formula A structure, R1 and R10 taken alone are hydrogen or halogen; R2, R5, R6 and R9 taken alone are hydrogen, alkyl, carboxyalkyl, aminoalkyl, alkylether, alkylthioether, halogen or alkoxy; R3, R4, R7 and R8 taken alone are hydrogen, alkyl, carboxyalkyl, aminoalkyl, cycloalkyl, aryl, or alkyl, cycloalkyl, or aryl substituted so as to have additional functional groups attached, including but not limited to alkoxy, sulfate, phosphate, or nitrate; R2 and R3 taken together are alkyl chains each having from 2 to 5 carbon atoms connecting the 2xe2x80x2 carbon to the nitrogen attached to the 3xe2x80x2 carbon; R9 and R8 taken together are alkyl chains each having from 2 to 5 carbon atoms connecting the 7xe2x80x2 carbon to the nitrogen attached to the 6xe2x80x2 carbon; R4 and R5 taken together are alkyl, each having from 2 to 5 carbon atoms connecting the 4xe2x80x2 carbon to the nitrogen attached to the 3xe2x80x2 carbon; R6 and R7 taken together are alkyl, each having from 2 to 5 carbon atoms connecting the 5xe2x80x2 carbon to the nitrogen attached to the 6xe2x80x2 carbon; R3 and R4 taken together form an alkyl or alkylene chain containing up to 5 atoms in the principal chain, consisting of carbon and one or more heteroatoms from the group consisting of nitrogen or oxygen, with both terminal valence bonds of said chain being attached to the nitrogen attached to the 3xe2x80x2 carbon; R7 and R8 taken together form an alkyl or alkylene chain containing up to 5 atoms in the principal chain, consisting of carbon and one or more heteroatoms from the group consisting of nitrogen or oxygen, with both terminal valence bonds of said chain being attached to the nitrogen attached to the 6xe2x80x2 carbon; R11, R12, R13, and R14 are each hydrogen or halogen.
One function of the Raxe2x80x2 substituent is to block lactam ring formation, and consequently it can be chosen from a wide variety of substituents such as alkyl, carboxyalkyl, aminoalkyl, cycloalkyl, aryl, or arylalkyl. The size of the substituent as a blocking group against lactam ring formation can vary considerably.
The linkage to desired conjugated substances is formed through the Raxe2x80x2 substituent or derivatives of the Raxe2x80x2 substituent. Typically one may select an Raxe2x80x2 that is alkyl, carboxyalkyl, aminoalkyl, cycloalkyl, arylalkyl, but preferably Raxe2x80x2 is chosen so as to include a chemically reactive functional group for further derivatization. Suitable functional groups are amines, alcohols, halogens, carboxylates, hydrazines, sulfhydryls, sulfates, phosphates, or nitrates. The Raxe2x80x2 substituent in its simplest form with a chemically reactive functional group may be xe2x80x94CH2CH2OH from which the hydroxyl may be used to prepare the desired various derivatives or conjugates.
Because compounds of the invention preferably possess functional groups linked through the 3-position carboxyl group the linkage converts the 3-position carboxylate to a non-acidic function (e.g. amide), which confers better stability to derivatives such as phosphoramidites. By virtue of doing the chemistry through the 3-position carboxyl group, the inventive dyes and labeling derivatives are single isomer forms, unlike compounds which require purification from mixtures of 5- and 6-position carboxyrhodamines before preparing oligonucleotide labeling reagents.
Since compounds of the invention possess a fully substituted amide nitrogen, the dyes are prevented from being converted to a non-fluorescent lactam form. Phosphoramidites and derivatized solid phase support matrix reagents of this invention allow efficient, fully automated synthesis of rhodamine labeled oligonucleotides.